Bacillus methanolicus MGA3 is a candidate for use as an industrial workhorse in methanol-based bioprocesses and its metabolism has been studied in systems biology approaches in recent years, including transcriptome characterization through RNA-seq. Here, we identify a putative lysine riboswitch in B. methanolicus, and test and characterize it. We also select and experimentally verify 10 putative B. methanolicus-derived promoters differing in their predicted strength and present their functionality in combination with the lysine riboswitch. We further explore the potential of a B. subtilis-derived purine riboswitch for regulation of gene expression in the thermophilic B. methanolicus, establishing a novel tool for inducible gene expression in this bacterium.Genome-wide transcriptomic data obtained in RNA-seq experiments can serve as a reliable source for identification of novel regulatory elements such as riboswitches and promoters. Riboswitches are parts of the 5′ untranslated region of mRNA molecules that can specifically bind various metabolites and control gene expression. For that reason, they have become an attractive tool for engineering biological systems, especially for the regulation of metabolic fluxes in industrial microorganisms. Promoters in the genomes of prokaryotes are located upstream of transcription start sites and their sequences are easily identifiable based on the primary transcriptome data.  In adenine-deficient conditions, the riboswitch is in an ‘off’-state and prevents gene expression by introduction of transcription termination through formation of a large hairpin [pbuE riboswitch can be used as a part of an inducible gene expression system. It can be activated by purine analogues that are close chemical variants of adenine, 2,6-diaminopurine , 2-aminopurine (2-AP), purine (P) and N6-methyladenine (MA) [Transcriptomic data are a source of ample information on different regulatory elements including putative riboswitches and promoters. Riboswitches are RNA  archaea . They ar archaea . Riboswi archaea . This ca purines . The mec purines ,5. TransNA decay ,7,8,9. A hairpin . This st hairpin . Thanks ine (MA) .Riboswitches have multiple potential uses in drug development as targets for antibacterial compounds, and in biotechnology as a tool for strain engineering. Popular engineering strategies involve the use of riboswitches to regulate the expression of the genes in the biosynthetic pathways competitive to the one of the chosen product ,12, to dUse of riboswitches for the fine-tuned regulation of gene expression is a promising prospect in strain engineering. In addition, access to repertoires of different promoters is fundamental for metabolic engineering of production strains. A common approach towards identification and characterization of novel promoters is based on screening of libraries of synthetic promoters, whose sequences are either completely randomized or partially based on consensus promoter sequences ,19,20,21Bacillus methanolicus is a thermophilic bacterium with the potential of becoming an industrial workhorse for biotechnological production of value-added chemicals [l-glutamate in methanol-controlled fed-batch fermentations, with final titers up to 60 g L−1 [B. methanolicus as a host organism has spiked the interest of the scientific community in this bacterium and led to developments in understanding its metabolism, including characterization of its transcriptome [B. methanolicus, novel regulatory elements, such as a mannitol-inducible promoter, were previously predicted and characterized [B. methanolicus, characterize it and use it in a gene regulation system. From the same data, we further identify and characterize 10 promoters of diverse strength. Moreover, we use previously described B. subtilis-derived pbuE riboswitch to control gene expression in B. methanolicus [hemicals . The wil60 g L−1 ,29. This60 g L−1 . The poscriptome ,33,34,35cterized . Here, wB. methanolicus were characterized through sequencing of mRNA isolated from B. methanolicus samples collected during cultivations in 16 different conditions [cis-regulatory RNA elements and novel transcripts.As previously described, the primary and whole transcriptomes of nditions . This alB. methanolicus encodes a putative amino acid permease similar to lysine permease YvsH from B. subtilis (51.64% identity), arginine:ornithine antiporter ArcD from Pseudomonas aeruginosa PAO1 (36.73% identity) and the lysine permease LysI from Corynebacterium glutamicum 13032 (27.77% identity). Rodionov et al. (2003) identified highly specific regulatory elements responsive to l-lysine upstream of the yvsH orthologs in different Bacillus species, e.g., B. subtilis, B. cereus, B. halodurans, Geobacillus steorothemophilus, which led them to suggest that YvsH is involved in the lysine transport in the above bacilli [It was predicted in that study that the 5′ untranslated region (5′ UTR) in mRNA belonging to the locus BMMGA3_01150 contains a putative lysine riboswitch. The gene located in locus BMMGA3_01150 in the genome of  bacilli . The seqHere, we study the transcript abundancies and the transcription start sites for respective transcripts to predict the sequence of the putative lysine riboswitch and to create a small set of promoters putatively differing in their strength, with a goal of detection and characterization of lysine riboswitches and novel promoters. Based on the available data for primary and whole transcriptome, 10 promoters differing in their strength were selected . The promdh) gene promoter (Pmdh) in pTH1mp-sfGFP construct was replaced with the 200 bp sequences identified upstream of TSS of the genes with loci listed in B. methanolicus hosts and the recombinant strains tested for fluorescence activity  or mdh promoter and B. methanolicus-derived lysine riboswitch (pTH1mplrBM-sfGFP), 20 mM l-lysine was added to the growth medium. A strain carrying the empty vector (pTH1mp) was used as control. The effect of l-lysine supplementations on the sfGFP fluorescence from different bacterial strains is shown in B. methanolicus, B. subtilis and E. coli), the sfGFP fluorescence decreased when l-lysine was added in the case where sfGFP expression is controlled by lysine riboswitch (pTH1mplrBM-sfGFP construct). For strains carrying the control vector (pTH1mp-sfGFP), the decrease in sfGFP fluorescence was either lower than for the strains carrying pTH1mplrBM-sfGFP or did not occur at all. Since the effect of l-lysine supplementation on sfGFP fluorescence in the strain carrying the control vector pTH1mp-sfGFP is visible only in one of the tested hosts, we attribute this effect to specific differences between the metabolism of the host organisms rather than to changes in the activity of mdh promoter. We additionally confirmed that the riboswitch responds exclusively to supplementation with l-lysine and no other amino acids tested . Some effect was shown due to supplementation of cadaverine; however, this was most probably due to the structural similarity between cadaverine and l-lysine  strain, that carries the plasmid contacting the construct with its native lysine riboswitch, in a minimal medium supplemented with the following concentrations of l-lysine 0, 0.2, 0.4, 2.5, 5, 10, and 20 mM. As shown in l-lysine. The 20 mM lysine is enough to fully inhibit expression of sfGFP in the tested strain.To assess the sensitivity of the l-lysine into the bacterial cells. To better understand the response of the riboswitch to different l-lysine intracellular concentrations, we analyzed the fluorescence of sfGFP reporter protein, encoded by a lysine riboswitch-controlled gene in the wild-type B. methanolicus MGA3 strain and a l-lysine-overproducing strain M168-20. Both strains were transformed with the pTH1mplrBM-sfGFP plasmid and the fluorescence levels in each strain were evaluated. B. methanolicus strain M168-20 is a l-lysine-secreting classical mutant able to produce 140 mg L−1 of lysine during shake flask cultivations, which is approximately 20-fold higher than the wild-type strain MGA3 with l-lysine titers of 7 mg L−1 during flask cultivations [sfGFP plasmid was cultivated with or without addition of l-lysine to the medium, no sfGFP fluorescence was observed, indicating that the intracellular l-lysine concentration was high enough to completely arrest the expression sfGFP under control of lysine riboswitch. The differences in l-lysine secretion between wild-type and lysine-producing strains were confirmed experimentally and with l-lysine titers for analyzed strains presented in l-lysine, and for regulation of gene expression and subsequent metabolic flux control in l-lysine-overproducing strains.An important consideration is that the response of the lysine riboswitch depends strongly on the import rate of ivations ,44,45. AB. methanolicus, we were able to identify a series of promoters differing in strength (mdh promoter in pTH1mp-sfGFP plasmid with the selected promoters. The sequence of the ribosome binding site (RBS) was left unaltered to exclude any effect of the protein translation on the final sfGFP fluorescence. A total of 10 promoters with predicted different strengths were selected for further testing. However, the sequence predicted to be the strongest chromosome-based promoter  was not included due to technical issues with strain construction. As expected, the use of the selected promoters for control of sfGFP expression led to different levels of sfGFP fluorescence (sfGFP), carrying the mdh promoter. However, since mdh is a plasmid-born gene in B. methanolicus, it is not possible to directly compare the transcriptomic data for that gene with the data on sfGFP fluorescence [By using the published transcriptomic data for rescence , either rescence .mdh promoter upstream of the lysine riboswitch. Based on the initial screening, four promoters were chosen to be used together with the lysine riboswitch for the control of sfGFP expression. Promoters p05 and p12 were included as promoters potentially stronger than mdh promoter, and promoter p18 as potentially weaker. We also included p01 (Phps-phi) which is predicted to be the strongest chromosomal promoter of B. methanolicus but was not able to experimentally validate this in this study. As shown in hps-phi promoter is 7.3-fold stronger than from mdh promoter however, it also leads to higher translation readthrough when the riboswitch is activated by addition of l-lysine. Some discrepancies are observed between the strength of tested promoters when the riboswitch is present in the construct in comparison to constructs without the riboswitch. However, the general trend for the promoter strength showed that p05 and p12 are stronger than the mdh promoter and p18 is weaker. Here, we could show that the riboswitch-controlled gene expression can be additionally affected by the strength of the promoter located upstream of this regulatory element.To expand the potential applicability of newly characterized lysine riboswitch and some of the tested promoters, we substituted the B. methanolicus the new genetic system for riboswitch characterization . The P1 region is a part of a purine riboswitch aptamer which forms a three-way RNA junction from helix P1 and hairpins P2 and P3, and changes in length of P1 helix affect the activity of pbuE riboswitch in the presence of 2-aminopurine [pbuE riboswitch under the control of the Phps-phi promoter.To further expand the genetic toolbox for rization  was usednopurine . AdditiopbuE riboswitch is functional in B. methanolicus, and it is activated by supplementation of 1 mM 2-aminopurine into the growth media. Furthermore, both exchange of the promoter as well as the introduction of a mutation in the P1 helix have effect on the activity of the pbuE riboswitch. Exchange of the mdh promoter to the Phps-phi led to two changes in the activity of the riboswitch. Firstly, the background fluorescence of the reporter protein, sfGFP, increased which can be caused the fact that the Phps-phi promoter is putatively stronger than the mdh promoter. Secondly, in an activated state of the riboswitch—the sfGFP fluorescence was higher in the construct with a Phps-phi promoter in comparison to mdh promoter, this observation is could also be caused by the differences in promoter strength. The modification in the P1 region led to increased responsiveness of the riboswitch to supplementation with 2-aminopurine and slightly increased background in comparison to the wildtype version of the riboswitch. The use of pbuE riboswitch with P1 modification leads to relatively low background expressions together with adequately high expression in its fully induced state. In fact, when the modified variant of the pbuE riboswitch was used, the sfGFP fluorescence in its fully induced state was as high as the sfGFP fluorescence in strain containing control construct pTH1mp-sfGFP. Altogether, we were able to create a novel system for control of gene expression in B. methanolicus.As shown in B. methanolicus was created and used for characterization of a putative native lysine riboswitch and a B. subtilis-derived pbuE riboswitch. Based on the available transcriptomic data for B. methanolicus MGA3 the sequence of the putative lysine riboswitch was annotated and experimentally validated. It was shown to be functional in its native host and in two heterologous, mesophilic hosts. To our knowledge, this is a first example of functional transfer of the riboswitch from a thermophilic host to its mesophilic counterpart.In this study, a genetic system for investigation of properties of riboswitches in the thermophilic and methylotrophic B. methanolicus-derived lysine riboswitch in its native host, l-lysine was supplemented to a final concentration of 5 mM (750 mg L−1) whereas B. subtilis and E. coli-derived riboswitches tested either in their native hosts or in C. glutamicum are activated by addition of 0.1 mM l-lysine or less [l-lysine can be caused either by differences in sensitivity of different riboswitches or by the activity of the l-lysine import system in different bacteria. The import and export system for l-lysine is not fully elucidated in B. methanolicus and it is, therefore, not possible to predict how the supplementation of growth medium affects the intracellular l-lysine concentrations in B. methanolicus [l-lysine-overproducing strain M168-20. Based on the available data for wild-type MGA3 and l-lysine-overproducing NOA2#13A52-8A66 strains, the intracellular l-lysine concentration in M168-20 can be assumed to lie within the range of 2.3–66.8 mM [l-lysine concentration is enough to fully suppress expression of sfGFP under control of lysine riboswitch in M168-20 strain without additional supplementation of growth media with l-lysine. This result suggest that the B. methanolicus-derived riboswitch is less sensitive to changes in l-lysine concentrations than E. coli and B. subtilis-derived riboswitches which may be caused by the fact that the intracellular concentration of l-lysine in B. methanolicus is higher than in B. subtilis or E. coli [In order to fully activate the  or less ,9,12. Thanolicus . For tha–66.8 mM . Our dat E. coli ,49.B. methanolicus in this study. Hitherto, only three promoters have been shown to be functional in B. methanolicus, one of them being mdh promoter. Methanol dehydrogenase constitutes up 22% of total soluble protein in cells of B. methanolicus grown under methanol-limiting conditions which makes it one of the most abundant proteins in the proteome of B. methanolicus [Furthermore, a set of 10 different promoter sequences were identified based on transcriptomic data and experimentally verified for anolicus ,50. The anolicus . Altogetanolicus ,24,25,26B. methanolicus. Three promoters tested in this study were chosen, together with the untested promoter that regulates the expression of the operon encoding 3-hexulose-6-phosphate synthase (Hps) and phosphohexose isomerase (Phi) Phps-phi (p01). Based on the transcriptomic and enzymatic activity data, this promoter can be assumed to be strong in B. methanolicus [mdh promoter with other promoters had, in most of the cases, a predictable effect on the sfGFP fluorescence intensity; the use of promoters designated as p01 and p12 led to increased sfGFP fluorescence when the lysine riboswitch was not activated and use of p18 led to decreased fluorescence in comparison to mdh promoter. The residual (background) sfGFP fluorescence after l-lysine supplementation differed accordingly depending on the promoter strength, as it was higher for stronger promoters. This finding shows the flexibility and predictability of the lysine riboswitch-controlled system which will allow for its future use in more complex genetic systems.Promoters from the newly created collection were combined with the sequence of the characterized lysine riboswitch to establish a new tool for regulation of gene expression in anolicus ,52. The B. subtilis-derived pbuE riboswitch was created and shown to be functional in B. methanolicus. This is in accordance with previous experiments, where it was active in different bacterial species, E. coli, B. subtilis, and thermophilic Geobacillus thermoglucosidasius and Clostridium thermocellum [B. megaterium-derived xylose-inducible promoter drives gene expression in B. methanolicus. Furthermore, signal peptides derived from G. stearothermophilus, B. subtilis and B. licheniformis support protein secretion in B. methanolicus [pbuE riboswitch-based gene expression system has beneficial features for use in B. methanolicus. Firstly, 2-AP that is used for its activation is a gratuitous inducer, meaning that it does not serve as a carbon source for B. methanolicus. Furthermore, a pbuE riboswitch-based gene expression system is titratable which renders it useful for sensitive applications, such as expression of host toxic gene products. The properties of the system can be modified through exchange of the promoter upstream of pbuE-riboswitch. However, use of a strong promoter Phpsphi- was not beneficial in this case as it led to increased background. The change in sequence of P1 helix, which functions as the antitermination element of the pbuE riboswitch, led to improved properties for regulation of gene expression, as the sfGFP fluorescence upon full induction increased in comparison to wild-type lysine riboswitch, while remaining tunable, inducible and maintaining a low background expression.Lastly, a system for regulation of gene expression based on the. mocellum ,54,55. Ianolicus ,56. The B. methanolicus, with lysine riboswitches being especially useful for regulation of the genes that belong to the lysine biosynthesis pathway and 2-aminopurine riboswitch for inducible gene expression. Furthermore, use of regulated gene expression systems that are functional both in E. coli and B. methanolicus can become a solution to reoccurring cloning problems caused by utilization of a strong, constitutive promoter Pmdh. High-level expression in E. coli is well studied and established, but expression of toxic genes especially requires special attention in order to avoid detrimental effects on the livability of E. coli cells [E. coli strains, reducing the copy number of ColE1-derived plasmids , or by reducing incubation temperature. In the case of the B. methanolicus-derived riboswitch described in this study, expression of the gene of choice is strongly reduced in E. coli when placed under control of the riboswitch, allowing toxic genes/proteins to be cloned.The riboswitch-based systems developed here can potentially be used for the regulation of gene expression in li cells . Widely li cells ,59,60. ME. coli strain DH5α was used as a general cloning host. The following strains were used as sources of gDNA for cloning of the lysine riboswitches: B. subtilis 168, B. methanolicus MGA3 and E. coli MG1655, and as hosts for screening of lysine riboswitches: B. subtilis 168, B. methanolicus MGA3. B. methanolicus M168-20 and E. coli DH5α.Bacterial strains and plasmids used in this study are listed in E. coli DH5α-competent cells were prepared according to the calcium chloride protocol, as described previously [B. subtilis 168, B. methanolicus MGA3 and E. coli MG1655 was isolated [B. methanolicus MGA3 and M168-20 were made electrocompetent and transformed by electroporation as described by Jakobsen et al. (2006) with modifications [eviously . All staeviously  or accorisolated . PCR proications ,66.E. coli and B. subtilis strains were cultivated at 37 °C in Lysogeny Broth (LB) or on LB agar plates [−1 chloramphenicol, respectively. For experiments involving lysine riboswitches, strains of E. coli and B. subtilis were cultivated at 37 °C in minimal medium MVcM supplemented with 10 g L−1 glucose, 20 mM l-lysine and 15 or 5 μg mL−1 chloramphenicol, respectively, unless stated otherwise. For standard cultivations, recombinant strains of B. methanolicus were cultivated in MVcM minimal medium with 200 mM methanol supplemented with 5 μg mL−1 chloramphenicol. MVcM medium contained the following, in 1 L of distilled water: K2HPO4, 4.09 g; NaH2PO4*H2O, 1.49 g; (NH4)2SO4, 2.11 g and was adjusted to pH 7.2 before autoclaving. MVcM medium was supplemented with 1 mL 1 M MgSO4*7H2O solution, 1 mL trace elements solution and 1 mL vitamin solution. The 1 M MgSO4*7H2O solution contained 246.47 g of MgSO4*7H2O in 1 L of distilled water, trace elements solution contained the following, in 1 L of distilled water: FeSO4*7H20, 5.56 g; CuSO4*2H2O, 27.28 mg; CaCl2*2H2O, 7.35 g; CoCl2*6H2O, 40.50 mg; MnCl2*4H2O, 9.90 g; ZnSO4*7H2O, 287.54 mg; Na2MoO4*2H2O, 48.40 mg; H3BO3, 30.92 mg; HCl, 80 mL and vitamin solution contained the following, in 1 L of distilled water: biotin, thiamine hydrochloride, riboflavin, calcium d-pantothenate, pyridoxine hydrochloride, nicotinamide, 0.1 g each; 4-aminobenzoic acid, 0.02 g; folic acid, vitamin B12 and lipoic acid, 0.01 g each [−1 xylose (v/v) was added for induction. The 20 mM l-lysine or 1 mM 2-aminopurine were added when necessary. For precultures, minimal medium supplemented with 0.25 g L−1 yeast extract, designated MVcMY, was used. Cultivations were performed in triplicates in 250 mL baffled flasks , inoculated to a starting OD600 = 0.2. Growth was monitored by measuring OD600 with a cell density meter (WPA CO 8000 Biowave). Specific growth rates were calculated from the exponential phase, by calculating the slope of semi logarithmic plots of optical density versus time over a suitable period of time.Recombinant r plates  suppleme1 g each . When ne−1 chloramphenicol and 20 mM l-lysine for at least 6 h, reaching a minimum OD600 = 0.4 (one doubling) before harvesting. For fluorescence microplate assays, cells were harvested by centrifugation at 13,000 rpm for 10 min and washed twice with PBS, before resuspension in PBS. A volume of 200 μL of resuspended cells were used for measuring fluorescence in microtiter plates . An Infinite 200 PRO plate reader (Tecan Group Ltd.) was used for fluorescence measurements, with settings: ex 485/9 nm, em 535/20 nm. sfGFP signals were collected with a gain setting of 90 and divided by OD600for normalization.Bacterial strains were cultivated at either 37 or 50 °C in MVcM supplemented with 5 μg mLFor the analysis of amino acids concentrations, 1 mL of the bacterial cultures was centrifuged for 10 min at 13,000 g. Extracellular amino acids were quantified by means of high-pressure liquid chromatography (Waters Alliance e2695 Separations Module) after FMOC-Cl (fluorenylmethyloxycarbonyl chloride) derivatization of the supernatants, according to the protocol described before . SamplesThe detection was performed with a fluorescence detector (Waters 2475 HPLC Multi Fluorescence Detector), with excitation and emission at 265 and 315 nm, respectively.B. methanolicus MGA3. Based on the RNA-seq data, we have detected a putative lysine riboswitch in the genome of B. methanolicus and confirmed its functionality through analysis of changes in sfGFP fluorescence with and without the presence of l-lysine. The response of the lysine riboswitch to changes in intra- and extracellular concentrations of l-lysine B. methanolicus demonstrates its feasibility for potential use in metabolic engineering for metabolic flux control or as a lysine sensor. Furthermore, ten novel promoters were predicted based on RNA-seq data and here experimentally shown to be functional in B. methanolicus. The promoters differ in their strength, ranging from 0.1- to 5.9-fold of the activity of the control promoter, Pmdh. A system for regulation of gene expression based on the B. subtilis-derived pbuE riboswitch was created and shown to be functional in B. methanolicus. Lastly, the genetic tools were used in combination, presenting the potential for creation of novel genetic systems for control of gene expression in B. methanolicus.In this report, we present the feasibility of riboswitch-based systems for control of gene expression in a thermophilic methylotroph,


Flexural strength, elastic modulus, fracture toughness, work of fracture, water sorption, and water solubility were evaluated. The 3D-printed test specimens were post-cured using two different units (Imprimo Cure® and Form Cure®). The tests were carried out after both dry and 30 days water storage. Data were collected and statistically analyzed. Resin type had a significant effect on the flexural strength, elastic modulus, fracture toughness, and work of fracture (p < 0.001). The flexural strength and elastic modulus for the heat-cured polymer were significantly the highest among all investigated groups regardless of the storage condition (p < 0.001). The fracture toughness and work of fracture of the 3D-printed material were significantly the lowest (p < 0.001). The heat-cured polymer had the lowest significant water solubility (p < 0.001). The post-curing method had an impact on the flexural strength of the investigated 3D-printed denture base material. The flexural strength, elastic modulus, fracture toughness, work of fracture of the 3D-printed material were inferior to those of the heat-cured one. Increased post-curing temperature may enhance the flexural properties of resin monomers used for 3D-printing dental appliances.The aim of this work was to investigate the effect of two post-curing methods on the mechanical properties of a 3D-printed denture base material. Additionally, to compare the mechanical properties of that 3D-printed material with those of conventional autopolymerizing and a heat-cured denture base material. A resin for 3D-printing denture base (Imprimo Complete dentures (CDs) are considered the standard treatment for the rehabilitation of complete edentulism ,2. Diffe3D-printing technology has been used to create a wide range of dental devices such as surgical guides, provisional crowns, dental splints, and denture bases ,9,10,11.Usually, the printed components are subjected to a post-curing procedure in an ultraviolet (UV) oven to achieve additional cross-linking of the monomer’s unreacted chemical groups and enhance the mechanical properties . The appTherefore, the aim of the present study was to investigate the effect of two different post-curing methods on the flexural strength, elastic modulus, fracture toughness, work of fracture, water sorption, and water solubility of a 3D-printed denture base material. Additionally, we aimed to compare the mechanical properties of that 3D-printed material with those of conventional autopolymerizing and heat-cured denture base material. The first null hypothesis was that different post-curing methods would not affect the investigated properties of the 3D-printed material, while the second null hypothesis was that all the investigated materials would have similar mechanical properties.® LC Denture; Scheu-Dental GmbH, Iserlohn, Germany), a heat-polymerizing , and an autopolymerizing acrylic resin , which is recommended by the manufacturer for removable partial dentures fabrication (® LC Denture is a light-curing methacrylate-based resin suitable for 3D printers with DLP technology (385 nm). Three test groups were designed for testing as follows: 3D-printed, Palapress, and Paladon. The 3D-printed group was further subdivided into two subgroups according to the post-curing device used: Imprimo Cure and Form Cure  containing a water-based cleaning agent , and then rinsed with isopropanol for additional 3 min, following the manufacturer’s instructions, in a separate device . Half of the 3D-printed test specimens were post-cured with the Imprimo Cure device , while the other half was post-cured with the Form Cure device . The support structures were removed after post-curing using low-speed rotary instruments (5000 rpm). The autopolymerizing and heat-cured acrylic resin test specimens were fabricated according to the manufacturer’ instructions, as described in a previous study [The 3D-printed specimens were virtually designed and then printed horizontally with a DLP 3D-printer . To remove fluid resin remnants from the test specimens, they were placed in ultrasonic cleaning equipment for 3 min  were tested for flexural strength and elastic modulus (n = 16/group). A static 3-point bending test was conducted in air using a universal testing machine , at a preload speed of 5 mm/min. PC software  was used to record the load-deflection curves and obtain the flexural strength and elastic modulus values. The distance, adjusted to provide support to the test specimens, was 50 mm.Sixty-four bar-shaped test specimens (10.0 × 65.0 ×3.3 ± 0.2 mmf) and flexural modulus (Ef) were calculated from the following formula:m is the applied load (N) at the highest point of the load-deflection curve, I is the span length, b is the width of the test specimens, and h is the thickness of the test specimens.Flexural strength (ơ3) were prepared (n = 12/group) to test the fracture toughness (KIc). The specimens were fixed on a flat holder, and a double-faced diamond disk  was used to make a 3.0 mm pre-crack in the center of each specimen under water-cooling. The pre-cracks were sharpened by a straight edged razor blade to make a notch with a depth of 100–400 µm. A light microscope  was used to check the crack length (10× magnification). The test specimens were submitted to a 3-point bending test on a universal testing machine at a crosshead speed of 1.0 mm/min. The specimens were placed on the supports of the test rig (32.0 mm distance) with the notch facing opposite the load plunger. After testing, 3 measurements of the notch length on the fracture surface of each specimen were recorded using the light microscope, and the average was computed and determined as the crack length (a). The KIc was calculated in MPa m1/2 using the subsequent mathematical statement [f) was calculated in J/m2 as follows [Δ is the recorded deflection for load P in newtons, B is the sample width in mm, H is the specimen height in mm, and a is the crack length in mm.Additional single-edge notched bend (SENB) test specimens KI follows :Wf = U/[1). The drying cycle was repeated until the discrepancy among consecutive weight measurements was lower than 0.1 mg. The specimens were submerged in 15 mL of distilled water/sample at 37 °C for 30 days after attaining a constant mass. The weight of the water-stored specimens was evaluated after 60 s of withdrawal from water and thorough drying with absorbent paper at 1, 2, 3, 7, 14, 21, 28, and 30 days. Using the same drying procedure as before, the specimens were reconditioned to a steady mass. The water sorption and solubility rate were calculated adopting the ensuing equations [1 is the dry mass (mg) of the sample afterwards storage in an air-drying device for 24 h, m2 is the mass (mg) of the specimen after water storage for 30 days, and m3 is the steady mass (mg) of the sample subsequent to the second drying cycle.In order to evaluate water sorption and solubility in percentage, forty circular test specimens (n = 10/group)  were tested. They were kept in an air-drying device involving dried silica at 37 ± 1 °C for 22 h and then at ambient laboratory conditions (23 ± 1 °C) for 2 additional hours. The test specimens were weighed on a scale  to an accuracy of 0.1 mg to get the values for the initial weight , while the effects of post curing method and storage conditions were detected using paired sample t-test and Wilcoxon rank-sum test. The significance level was set at p < 0.05.All data for the evaluated properties were collected and statistically analyzed with a statistical software program . Due to nonhomogeneous variances, Welch’s ANOVA (robust one-way ANOVA) was used to compare data between different groups, while pairwise comparisons of the groups were performed by Dunnet’s T3 post hoc test. Welch p < 0.001) and water sorption (p = 0.004) on the investigated materials. Only flexural strength was significantly affected by the storage condition (p = 0.001). The effect of post-curing method was significant on the flexural strength of the 3D-printed specimens (p = 0.021).The type of resin had a significant effect on the flexural strength, elastic modulus, fracture toughness, work of fracture . However, a significant reduction was seen in both properties after water storage (p < 0.05). A non-significant difference (p > 0.05) was noticed among the dry samples of the other groups in terms of the flexural strength and elastic modulus values. However, after water storage, the Imprimo Cure® subgroup recorded the lowest strength and modulus, which were significantly lower than Paladon® (p < 0.001).The mean values for the flexural strength and elastic modulus of the tested groups are presented in p < 0.001) regardless of the storage condition. Water storage resulted in a significant increase in fracture toughness and fracture work values for Palapress .p < 0.001) . The water solubility for Paladon® was significantly the lowest among the groups (p < 0.001). A statistically significant difference was found between the water solubility values of the Imprimo Cure® and Form Cure® subgroups (p < 0.001).Water sorption and solubility values were significantly different between groups (< 0.001) . Figure In this in vitro study, the effect of two different post-curing methods on the mechanical properties of a 3D-printed denture base material was investigated. Those investigated mechanical properties were flexural strength, elastic modulus, fracture toughness, work of fracture, and water sorption and solubility. Additionally, a comparison of those mechanical properties was made between conventional and 3D-printed resins. The findings revealed that the post-curing method had a significant effect on the flexural strength of both the dry-stored samples, and the water solubility of the 3D-printed resin material. Furthermore, substantial differences between the characteristics of 3D-printed and conventional denture base materials were found. As a result, the first null hypothesis was partially rejected, while the second hypothesis was totally rejected.DLP is one of the most advantageous 3D-printing technologies for dental applications due to its quick processing speed, superior resolution, and reasonable cost of the printer and its components ,14,29. DThe flexural strength of a material is defined as the maximum bending stress that can be applied to that material before it yields. Denture bases are prone to fracture when subjected to static or dynamic loading . Therefo® device, may elucidate the post-curing method’s significant effect on the flexural strength and water solubility of the 3D-printed material. Light intensity and temperature, for instance, have a considerable effect on the degree of double bond conversion and polymer characteristics [The combined effect of photo and thermal polymerization, as well as the extended post-curing duration within the Form Cureeristics ,37,38. Ieristics ,36,38,39eristics . Anothereristics  was thatWater uptake by resin materials is a diffusion-controlled process, which occurs either through its penetration into empty space such as micro-voids, or by a particular molecular interaction ,41. The According to the findings of this study, clinicians should consider the differences in mechanical characteristics between conventional and the 3D-printed materials used for denture base construction. Further improvements in the 3D-printed resin materials’ properties by composition modification or reinforcement are still needed. The correct selection of post-curing method could be an option for improvement.This study was limited by the investigation of only one type of 3D-printed denture base material and only two post-curing units. Furthermore, the produced specimens did not replicate the denture configuration. Further research investigating various 3D-printed materials and post-curing methods is recommended.The post-curing method has an impact on the flexural strength of the investigated 3D-printed denture base material. Increased the post-curing temperature may enhance the flexural properties of resin monomers used for the 3D-printing of dental appliances.Higher water solubility and inferior mechanical properties were found on the 3D-printed material when compared to the heat-cured one. The high temperature and extended processing time used for the heat-cured polymers might be attributed to their reduced water sorption and solubility.According to the findings of this study, the following can be concluded:


Purpose: Most therapeutic agents have limitations owing to low selectivity and poor solubility, resulting in post-treatment side effects. Therefore, there is a need to improve solubility and develop new formulations to deliver therapeutic agents specifically to the target site. Gelatin is a natural protein that is composed of several amino acids. Previous studies revealed that gelatin contains arginyl-glycyl-aspartic acid (RGD) sequences that become ligands for the integrin receptors expressed on cancer cells. Thus, in this study, we aimed to increase the efficiency of drug delivery into cancer cells by coating drug-encapsulating liposomes with gelatin . Methods: Liposomes were coated with gelatin using electrostatic interaction and covalent bonding. GCLs were compared with PEGylated liposomes in terms of their size, zeta potential, encapsulation efficiency, stability, dissolution profile, and cell uptake. Results: Small-sized and physically stable GCLs were prepared, and they showed high drug-encapsulation efficiency. An in vitro dissolution study showed sustained release depending on the degree of gelatin coating. Cell uptake studies showed that GCLs were superior to PEGylated liposomes in terms of cancer cell-targeting ability. Conclusions: GCLs can be a novel and promising carrier system for targeted anticancer agent delivery. GCLs, which exhibited various characteristics depending on the coating degree, could be utilized in various ways in future studies. There are several types of lipid-based nanoparticles used as drug delivery systems, such as liposomes, lipid nanoparticles, and solid lipid nanoparticles. One such example is liposomes, which are a type of nanocarrier consisting of phospholipids and cholesterol. Liposomes can encapsulate both hydrophobic and hydrophilic drugs owing to their amphipathic properties, and the surface of liposomes can be modified using functionalized lipids ,2. The pCoating can enhance the physical stability, targeting ability, and biocompatibility of nanoparticles and reduce their toxicity. Gelatin is a natural protein that is composed of several amino acids. A recent study revealed that gelatin contains arginine, glycine, and aspartate (RGD) sequences ,14. ThesIn this study, the hydrophobic molecule paclitaxel (PTX) as a drug or rhodamine B (RhoB) as a model payload was used. PTX is an anticancer drug used for treating various types of solid tumors and is known to inhibit tumor growth by preventing the breakdown of microtubules during cell division . As PTX The purpose of this study was to prepare gelatin-coated liposomes (GCL) and further characterize their stability and cell uptake. Therefore, GCLs were prepared and compared with PEGylated liposomes in terms of size, surface charge, encapsulation efficiency (EE), physical stability, dissolution profile, and cell uptake. As shown in Hydrogenated soybean phosphatidylcholine  and N-(carbonyl-methoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine  were purchased from Lipoid GmbH . 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-hydroxysuccinimide (DSPE-NHS) was bought from Nano Soft Polymers Co., Ltd., . Cholesterol, stearylamine, and gelatin from bovine skin  were purchased from Sigma-Aldrich . PTX was purchased from Tokyo Chemical Industry Co., Ltd. . RhoB was obtained from Sigma-Aldrich . The Float-A-Lyzer G2 (molecular weight cut-off (MWCO): 100 kDa) was purchased from Spectrum Laboratories, Inc. The Pur-A-Lyzer Mini (MWCO: 12 kDa) was purchased from Sigma-Aldrich . The Bradford protein assay kit (Pierce Coomassie Protein Assay Kit) was purchased from Thermo Scientific . Milli-Q quality purified water  was used throughout the study. All organic solvents were purchased from Samchun Chemicals . All other chemicals were of reagent grade. The NanoDeBee homogenizer  was used for the preparation of liposomes.Liposomes were prepared by referring to several existing studies on thin-film hydration methods ,20. Briev/v) as the mobile phase. An injection volume of 20 μL of PTX was used, and it was detected at a wavelength of 227 nm. The concentration of PTX was calculated from a calibration curve constructed using solutions containing varying concentrations of PTX (0.01–100 μg/mL).The PTX content in liposomes was determined using high-performance liquid chromatography (HPLC) based on the standard curve described in published articles ,22,23. TThe EE of each formulation was calculated using the following equation:v/v) ratio of liposome solution to gelatin solution and incubated at 25 °C and 50 rpm for 18 h. For GCL-2, the N-hydroxysuccinimide (NHS) liposome solution (pH 7.0) was mixed with two different amounts of the gelatin solution (pH 8) and incubated at 25 °C and 50 rpm for 24 h. After that, to remove free gelatin, the solution was dialyzed in a membrane tube (MWCO 100 kDa) in PBS (pH 7.4) for 4 h. The gelatin-coating efficiency was measured using a Bradford protein assay [The prepared plain liposomes were coated with gelatin in phosphate-buffered saline (PBS) (pH 8.0) via electrostatic interaction (GCL-1) or covalent bonding (GCL-2). For the GCL-1, the liposome solution was mixed with various concentrations of a gelatin solution  in a 1:1 (m) and melting point.A DSC system  was used to characterize the phase transition behavior of the materials and liposomes. Briefly, 3–5 mg of lyophilized liposomes were placed in standard aluminum pans. The measurements were performed in the temperature range of 0 to 200 °C, with a heating and cooling rate of 10 °C/min. A blank pan was used as a reference. The TA Universal analysis software was utilized to identify the phase transition temperature  at 4 cmThe stability of the GCLs was determined via size measurements using Dynamic Light Scattering (DLS). To analyze the time-dependent physical stability, the sizes of the PEGylated liposomes, GCL-1, and GCL-2 were measured at 4 °C and 25 °C for 8 weeks in PBS with 0.5% Tween 80. Before each measurement, the samples were shaken by inverting the tube. The stability of the GCLs was compared with that of the PEGylated liposomes.w/v) Tween 80 and incubated at 37 °C with the rotation at 50 rpm. The dissolution medium (1 mL) was collected at various time points  and replaced with 1 mL of a fresh medium at 37 °C. The amount of released PTX was determined using HPLC.The in vitro release profile was investigated using previously reported methods with minor modifications ,23,25. A2 at 37 °C. In the cell-uptake study, to analyze liposome uptake by the cells, RhoB (a fluorescence agent) was used instead of PTX because of their similar properties. To assess the cellular uptake of free RhoB or RhoB-loaded liposomes, RhoB was added to the formulation at a 1% molar ratio of the lipid. The cells were seeded in 96-well plates at a density of 2 × 104 cells/well in the culture medium (100 μL) and incubated for 24 h. After 4 h of treatment with 10 mg/mL of free RhoB, RhoB-loaded PEGylated liposomes, or RhoB-loaded GCLs, the cells were rinsed twice with Dulbecco’s phosphate-buffered saline (DPBS) and detached. To determine the cellular uptake of RhoB-GCLs, the fluorescence signal of the RhoB was measured using a microplate reader  linked to a fluorescence detector (578 nm).HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (2 g/L for RPMI-1640 and 4.5 g/L for DMEM) and 1% penicillin/streptomycin in humidified air with 5% COp < 0.05 was considered to be statistically significant.All of the studies were carried out in triplicate, and the results were expressed as mean ± standard deviation (S.D.). The statistical significance of the data was analyzed using the Student’s t-test. In all cases, g) of the phospholipids. After sufficient hydration, large-sized multilamellar vesicles (MLVs) were formed in the liposome dispersion. For intravenous injection (IV), the size of the liposomes should be small and uniform. Thus, the liposome dispersion was extruded 10 times through the NanoDeBee to reduce the size of the liposomes. The EE of liposomes is an important indicator for clinical applications. Usually, liposomes with EEs of more than 80% of the drug are considered effective [Three liposome formulations  were prepared using the film-forming and hydration methods. GCL-1 showed a positive charge due to stearylamine, whereas GCL-2 showed a negative charge. Hydration was performed at 70 °C above the glass transition temperature  as a solvent . For GCLThe size, polydispersity index, and zeta potential of the liposome formulations are shown in Particles with zeta potentials above30 mV or under -30 mV have an electrical repulsive force, thus decreasing their tendency to aggregate and increasing their stability in the colloidal state . PEGylatg and melting point of each substance, as measured using DSC. Melting peaks were observed at 147.96 °C for cholesterol and 85.66 °C for HSPC. The peaks of Tg for DSPE-NHS and DSPC-PEG2000 appeared at 56.86 °C and 53.88 °C, respectively, but the Tg of HSPC was not obtained owing to low calories.g of liposome components using DSC is critical. Tg is the temperature at which the transition occurs from the ordered gel phase to the lipid’s physical state. As the temperature in the hydration step must be higher than the Tg of the lipids, the experiment was performed at 70 °C.Measurement of the T2–CH2 bond was observed at 2850 and 2917 cm−1, and a peak corresponding to the C=O bond was observed at 1735 cm−1. Additional peaks corresponding to C–H, C–O, and N–H were observed at 1571, 1386, 1232, and 1055 cm−1, respectively. As gelatin was coated on the surface of the liposomes (GCL-1 and GCL-2), a new wide peak at around 3300 cm−1 appeared that corresponded to hydroxyl and amide bonds  in the gelatin moiety, confirming that the gelatin coating was on the liposomes. In addition, peaks for C=O, N-H, C-H, C-O, and C-C were observed at 1735, 1571, 1467, 1386, 1232, and 1055 cm−1, respectively. In the case of GCL-2 (−1 corresponding to the carbonyl group (C=O) of the amide bond was observed, indicating the successful coating of gelatin on the liposome.of GCL-2 B, which To evaluate the stability of the liposomes, the particle size of each formulation was measured for 8 weeks using DLS at 4 °C and 25 °C . The PEGw/v) was added to PBS, PTX solubility increased to 17.19 μg/mL. However, 0.1% of Tween 80 (w/v) in PBS was sufficient to create a sink condition, which is a dissolution media volume that has the ability to dissolve at least 3-5 times the amount of payload than the saturation volume. Because the molecular weight of PTX is 853.906 Da, dialysis tubes with an MWCO of 12 kDa were used.Prior to the release study, to determine the amount of soluble PTX in the release medium, the solubility of PTX in various solvent concentrations (Tween 80 in PBS) was measured to determine sink conditions. The results are shown in As shown in To enhance the targeting ability of liposomal carrier systems, we developed GCLs in this study. The gelatin coating on the liposomes worked as intended, and the GCLs exhibited different properties depending on the coating mechanism, namely electrostatic interaction or covalent bond formation. The small-sized formulations of GCLs  were stable for 8 weeks at 4 °C and 25 °C. Gelatin was originally used to increase the affinity of the liposomes to cancer cells, but it also conferred on the liposomes a sustained drug-release property. Gelatin appeared to increase the physical stability of the liposomes by acting as a protective colloid. The ability to control the drug-release pattern according to the degree of coating offers many possibilities. Cell-uptake studies showed the superior cancer cell-targeting ability of GCLs over PEGylated liposomes. This aspect of GCLs could allow for the selective delivery of a payload drug into cancer cells after intravenous injection into the bloodstream. The mechanism for efficient cell uptake of GCLs could be explained on the basis of receptor-mediated endocytosis through the RGD sequences and integrin receptors in HeLa cells. These results should be confirmed in more detail using in vivo studies. Although coating liposomes with natural proteins (like gelatin) is an alternative candidate to PEGylation, there are some limitations to direct commercialization due to their immunogenicity, manufacturing complexity, and cost. Therefore, further studies should focus on overcoming these limitations.


Transarterial chemoembolization (TACE) is an effective locoregional therapy in hepatocellular carcinoma (HCC). However, it is difficult to predict the tumour response (TR) of TACE intraprocedurally. The aim of this study was to predict the TR after TACE (1–3 months) in HCC patients using intraprocedural intraarterial contrast enhanced ultrasound (IA-CEUS).In this case-control study, consecutive patients who received TACE in our hospital from September 2018 to May 2019 were enrolled. IA-CEUS was performed before and after TACE. Postoperative contrast-enhanced liver MRI was performed 1–3 months after TACE as the gold standard. According to the modified Response Evaluation Criteria in Solid Tumours (mRECIST), ultrasonic manifestations were compared between the complete remission (CR) group and non-CR group by univariate and multivariate analyses. A logistic predictive model was established and validated, and its diagnostic efficiency was evaluated.P = 0.020), a larger dimension of non-enhancing area in superior mesenteric artery (SMA)-CEUS than the size in B-mode ultrasound preoperatively , presence of corona enhancement in hepatic artery (HA)-CEUS postoperatively , and decreased corona enhancement thickness (per centimetre) postoperatively . The area under the receiver operating characteristic curve (AUROC) of the predictive model was 0.904 . The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were 81.08, 91.67, 85.25, 93.75, and 75.86%, respectively. Leave-one-out cross-validation (LOOCV) showed that the accuracy was 77.05%.Forty-four patients with sixty-one lesions were enrolled in the study. Multivariate analysis identified, the risk factors as a large lesion diameter (OR: 1.84; 95% confidence interval [CI]: 1.009, 3.080; Intraprocedural IA-CEUS can be used to predict the TR in HCC patients after TACE. Liver cancer is the sixth most common cancer and the third most common cause of death. Hepatocellular carcinoma (HCC) accounts for 75–85% of liver cancer . TransarThe significance of the intraprocedural evaluation and prediction of TR lies in their ability to help to determine the end-point of TACE, and thus help determine future treatment and predict prognosis, which is beneficial to patients , 8. HoweAs a pure blood-pool imaging method, contrast-enhanced ultrasound (CEUS) is a highly accurate method for displaying tissue microcirculation in real time, providing higher resolution in the microcirculation than DSA, CT and MRI –19. It cThis single-centre study was approved by the Ethics Committee of the Chinese People’s Liberation Army General Hospital . All the patients provided written informed consent.Consecutive patients who received TACE treatment at PLA General Hospital from September 2018 to May 2019 were enrolled in this study.The inclusion criteria were as follows: (a) the patient met the diagnostic criteria of the European Association for the Study of the Liver ; (b) theThe clinical characteristics of the patients, including sex, age and hepatitis background, were recorded before TACE.All TACE procedures were performed by two experienced interventional radiologists (with 15 and 5 years of TACE experience) in a consistent way. After local anaesthesia, the right femoral artery was punctured with the modified Seldinger technique. A 4-Fr arterial catheter  was inserted for selective HA and SMA, and then a 3-Fr microcatheter  was superselectively inserted into the supply artery of the tumour. An oil-in-water-based emulsion comprising chemotherapy drugs epirubicin (30–50 mg), oxaliplatin (100–150 mg), 5-fluorouracil (5-FU) (500–750 mg) and leucovorin (200–300 mg) mixed with lipiodol (8 ml) at a proportion of 1:1 were injected through a microcatheter, and then 0.1 g polyvinyl alcohol (PVA) particles (300–500 μm) and gelatine sponge particles were injected successively. The treatment endpoint was determined by the interventional radiologist, and the main manifestation was the complete coverage of the tumour site by lipiodol deposition and feeding artery shown as a “dry-branch”, which means there were no blood supply absolutely.All US examinations were performed by one experienced US physician  using a Siemens Oxana2 or S2000 ultrasonic system equipped with CEUS software. The transducer was a C6–1 convex array transducer operated at 3.5–5.0Mz. All of the parameters were adjusted to ensure high-quality images. Before TACE, B-mode US was performed to define quantity (single or multiple), diameter (measured on the maximum section), location (lobe), and echogenicity  of the lesions. The gain, depth, focus, and dynamic range were appropriately adjusted. Although each patient had multiple nodules, specific nodules were selected for observation. The inclusion criteria for the nodules were (a) a diameter less than 10 cm; (b) a depth of no more than 8 cm from the skin; and (c) a good acoustic window for clearly display by US.IA-CEUS, including hepatic artery contrast-enhanced ultrasound (HA-CEUS) and superior mesenteric artery contrast-enhanced ultrasound (SMA-CEUS), was performed using an arterial catheter inserted before TACE, while HA-CEUS was performed again immediately after TACE. The time interval between IA-CEUS and TACE was less than 10 min. During HA-CEUS, the contrast agent reached the tumour directly through the supplying artery. There was no contrast agent in the portal vein, so the tumour only had enhancement of the arterial phase. In SMA-CEUS, the contrast agent reached the tumour through the portal vein, so the portal vein supply of the tumour was shown alone without disturbance of the arterial phase.Contrast pulse sequence mode was selected for image acquisition. Before performing CEUS, the lesions were reconfirmed in B-mode US. The mechanical index was set in the range of 0.06 to 0.1. The focus was set at the bottom of the screen. The gain and depth were adjusted accordingly. The contrast agent was sulphur hexafluoride . The contrast agent for SMA-CEUS was dissolved according to the specification , and the bolus injection dose was 1.5 ml. The concentration of contrast agent in HA-CEUS was 0.02 ml/ml , and the injection dose was 4 ml (at a speed of 1 ml/second). Continuous storage of the dynamic images spanned no less than 3 min.After TACE and IA-CEUS, IA-CEUS images were retrospectively evaluated frame-by-frame offline by two experienced US physicians . An image was excluded if it was considered to be of poor quality by both doctors. When there was a disagreement between them, they discussed it together until a consensus was reached.Parameters observed in HA-CEUS before and after TACE included intratumoural enhancement (presence or absence), corona enhancement in the late phase (presence or absence) and corona thickness (measured at the thickest point). The decrease in corona thickness after TACE was calculated. The parameters observed in SMA-CEUS before TACE included intratumoural enhancement (presence or absence) and the dimension of non-enhancing area (compared with B-mode ultrasound in the same section). The result of dimension of non-enhancing area can be divided into larger non-enhancing area and no larger non-enhancing area. Examples can be seen in Fig. Contrast-enhanced liver MRI was performed as the gold standard of TR for all patients to evaluate whether the nodules had a blood supply 1–3 months after TACE. Images were analysed by two abdomen MRI radiologists (with more than 10 years of experience) who were blinded to the IA-CEUS results. The result of TR in this study was evaluated on the bases of nodule rather than patient. Lesions were divided into a complete remission (CR) group and a non-CR group according to the modified response evaluation criteria in solid tumours (mRECIST) standard , 5. CR w2 tests were used to analyse differences. After univariate analyses, variables with statistical significance were subjected to logistic regression. P < 0.05 was considered statistically significant. The logistic predictive model was established based on the multivariate analysis and the Hosmer-Lemeshow goodness-of-fit test and leave-one-out cross-validation (LOOCV) were performed to validate the model. The receiver operating characteristic (ROC) curve was obtained, and the sensitivity, specificity, accuracy and area under the ROC curve (AUROC) were calculated. The data were analysed by using the statistical software SPSS 21.0 .All data were separately recorded by two researchers (Z.L.H. and H.P.), and then the accuracy was evaluated. For data following a normal distribution, continuous variables were expressed as mean values and standard deviations, and the independent t-test was used to analyse differences. For data following a nonnormal distribution, continuous variables were expressed as medians and interquartile ranges. and the Mann-Whitney U-test was used to analyse differences. Categorical variables were represented as frequencies and percentages, and independent or modified χAs presented in Table For HA-CEUS and SMA-CEUS, the observers obtained identical results in terms of intratumoural enhancement, and judgment of whether the dimension of non-enhancing area in SMA-CEUS was larger than the size in B-mode ultrasound. The kappa value was 0.902 for the evaluation of corona enhancement for HA-CEUS before and after TACE.P = 1.00).For HA-CEUS, all the lesions showed hyperenhancement before TACE, while only four lesions showed the presence of intratumoural enhancement in SMA-CEUS. There were no statistically significant differences between the two groups in these two parameters (P < 0.001). There was no significant difference in age (P = 0.18), sex (P = 0.61), hepatitis background (P = 0.41), nodule location (P = 0.94) or echo(P = 0.85) between the two groups.As shown in Table P = 0.046). There were no significant differences in corona enhancement (P = 0.14) or corona thickness (P = 0.31) between the two groups before TACE.Among the parameters of preoperative IA-CEUS, in the incomplete remission group, the non-enhancing area of SMA-CEUS was more likely to be larger than the size of the tumour shown in B-mode US , corona enhancement (P = 0.004) , and the decrease in corona enhancement thickness (P = 0.006) showed significant differences between the two groups.After TACE, intratumoural enhancement ( P = 0.00 (shown iP = 0.020) risk of a poor response. Preoperatively, tumours with an larger non-enhancing area in SMA-CEUS had a 3.379-fold  risk of a poor response. Postoperatively, the presence of corona enhancement in the late phase was associated with a 6.642-fold  risk of a poor response, and this risk was reduced by 6.8%  for every 1 cm reduction in the corona enhancement.Due to the clinical association between the presence of corona enhancement and corona thickness after TACE, the latter was excluded from the logistic regression. Table p < 0.001). The ROC curve is shown in Fig. P = 0.91) indicated that the model fit the data adequately. LOOCV was performed to verify the diagnostic efficacy of the model, and the results showed that the model accuracy was 77.05% (47/61).A logistic predictive model was established, and the AUROC was 0.909  guidelines, the clinical application of CEUS was expanded in this study. Although most of the contrast agents were used intravenously in previous studies, contrast agents were used in this study for imaging in the blood pool and did not cause adverse reactions. A similar method was used in a previous study to observe the microcirculation of tumours .


Assessment the impact of disability on mortality among the elderly is vital to healthy ageing. The present study aimed to assess the long-term influence of disability on death in the elderly based on a longitudinal study.This study used the Chinese Longitudinal Healthy Longevity Study (CLHLS) data from 2002 to 2014, including 13,666 participants aged 65 years and older in analyses. The Katz ADL index was used to assess disability status and levels. Cumulative mortality rates were estimated by the Kaplan-Meier method. Cox proportional hazards models were conducted to estimate associations between disability and all-cause mortality for overall participants, two age groups as well as specific chronic disease groups. All reported results were adjusted by survey weights to account for the complex survey design.P = 0.029) and level G . For patients with hypertension, diabetes, heart disease, cerebrovascular disease as well as dementia, disability increases their relative risk of mortality by 1.64 , 2.85 , 1.45 , 2.13  and 3.56  times, respectively.During the 12-year follow-up, the death density was 6.01 per 100 person-years. The 3-years’ cumulative mortality rate of nondisabled elderly was 11.9% . As the level of disability increased, the cumulative mortality rate was from 28.1%  to 77.6% . Compared with non-disabled elderly, the multiple-adjusted hazard ratio of death due to disability was 1.68 . The hazard ratios varied from 1.44  to 4.45  after classifying the disability levels. The hazard ratios of death in the young-old group (65–79 years) were higher than the old-old group (80 years and over) in both level B (HR = 1.58, 95%CI: 1.25, 2.00 vs. HR = 1.22, 95%CI: 1.06, 1.39, Disability increases the risk of all-cause death in the elderly, especially those with chronic diseases and the young-old group. Further studies are needed to better understand how to effectively prevent disability in the older population.The online version contains supplementary material available at 10.1186/s12877-021-02611-1. Ageing is a worldwide issue and presents tremendous challenges. The World Population Ageing 2020 Highlights  showed tPrevious studies showed that disability in the elderly was higher and inevitable at the end of life –8. The FHowever, there is a lack of evidence related to exploring the effects of disability status on death in the Chinese elderly. Besides, some studies used a dichotomous (yes or no) representing disabilities in activities of daily living , 7–9, igData was obtained from the CLHLS, a nationwide longitudinal study aimed at understanding factors related to healthy longevity in older Chinese population. Details of the study design, survey methodology, response rates and quality assessments of the data have been described in previous studies , 13.The samples were randomly selected from 22 of 31 provinces in China, representing approximately 85% of the Chinese population. Eight waves of surveys were implemented in 1998, 2000, 2002, 2005, 2008, 2011, 2014 and 2018. In the 1998’s survey, CLHLS attempted to interview almost all centenarians. Researchers recruited approximately equal numbers of participants aged 80 years and over by random sampling design. Over-sampled were used to avoid the problem of small sub-sample sizes at the more advanced ages, especially for oldest-old males. In the 2002’s survey, the CLHLS added a sub-sample of older adults aged 65–79 in the same area . All infN = 266) because of lack of sampling weights, had missing data in death date (N = 91), died at baseline year (N = 44) or lack of at least once interview in 2005, 2008, 2011 or 2014 (N = 1997). There were 13,666 participants (4133 young-old aged 65–79 years and 9736 old-old aged 80+) included for analysis , education , place of residence  and co-resident status . Lifestyle covariates included smoking  and alcohol drinking . Blood pressures were measured after sitting for 5 min. Hypertension was defined as systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg or had a self-reported history of hypertension. Other chronic disease histories included diabetes, heart disease, Parkinson’s disease, dementia, cerebrovascular diseases and cancer were self-reported.The CLHLS study was approved by the Research Ethics Committee of Peking University and Duke University (IRB00001052–13074). The informed consent was signed by the subject or a close relative . All metThe characteristic of participants was strata by two age groups (65–79 yeas versus 80 years and over). Considering the over-sampled design approach in the CLHLS study, Sample weights were used to illustrate the complex survey design in all analyses , 19. Prow was the age(x), sex(s), urban-rural residence(r) special weights provided in the CLHLS; Tj as the total number of interviewed persons of age group j; the n was the number of persons of age x, sex s, and residence r.t-test. Categorical variables were described as percentages (%) and compared using the Rao-Scott chi-square test [z statistic was used to test the difference in HRs between the age-group Cox models [Continuous variables were described as weight-adjusted means (± standard deviation [SD]) and compared using are test . Cumulatare test  were usex models . BesidesP < 0.001) during 12 years of follow-up.During the study period, 9, 950 (72.81%) out of the 13, 666 older adults died. The median follow-up period was 4.8 ± 3.8 years . The overall death density of the elderly was 6.01 per 100 person-years . The disability rate was 5.62% in the 65–79 age group and 21.21% in the old-old age group (P < 0.001). The distribution of disability levels (Katz index group) also statistically significantly differed between age groups (P < 0.001). The Kaplan-Meier survival curve . In particular, 20.73% of the young-old adults with disabled survived to the end of the 12-year follow-up period, while this proportion was only 5.51% in the old-old group with disability.The characteristics of the subjects were described in Table P = 0.189). However, the hazard risk of death was higher among the young-old elderly when one of six ADL functions (P = 0.029) or all six ADL functions loss (P < 0.001).Table P < 0.05). Specifically, disability raised hypertension patients’ death risk by 1.64  times, diabetes patients’ by 2.85  times, heart disease patients’ by 1.45  times, cerebrovascular disease patients’ by 2.13  and 3.56  times in dementia patients.Figure The key findings from this prospective study of old Chinese were as follow: (1) accompanying disability in ADLs had a strong impact on mortality rates raise in both the young-old and old-old groups. (2) Compared with the old-old group, a higher death risk was observed among the young-old group when the elderly had one of six or all six ADLs functions disabled. (3) For the elderly who suffered from chronic diseases, disability further increased the risk of death.Consistent with previous studies , we founP < 0.001). It indicated that there was about 4 of every 100 elderlies can avoid death each year if effective prevention measures on disability were implemented.Our study showed that even if only one of the daily activities was impaired, the 3-years cumulative mortality in the elderly was as high as 28.1% . Even in the young-old group, the 3-years cumulative mortality rate reached 22.7% . Unfortunately, Thomas M. Gill’s study [The explanation for the correlation that disability in the elderly increased the risk of all-cause dying is multifactorial. On the one hand, age-related frailty was an important factor in physical disability and mortality , 24, 25.A population-based cohort study with more than 10-years’ follow-up  showed tThis study had both strengths and limitations. The strengths included: (1) we detailed the classification of ADL disability by number and components to assess the impact on mortality after adjusting sampling weights, which had rarely been taken into account in previous reports despite its importance for subjects with disability and significantly overexposed to death risk. (2) Moreover, we additionally analyzed the impact of disability on mortality in older adults with common chronic conditions, which had important implications for targeted preventive measures in primary care. 3) Finally, the sample of incident cases of disability was population-based and covered a 12-year follow-up period. There were limitations to be acknowledged. Underreporting was unavoidable in the self-report questionnaire studies, which led to misclassification of chronic disease and disabling levels in some populations. Although the Cox model adjusted for a number of variables, there were inevitably unobservable confounding factors. Therefore, the findings needed to be treated with caution. Although the CLHLS study design used weighted sampling, the original study design focused on the oldest-old and the sampling of the young-old group was insufficient. However, it was no denying that the demographic characteristics of our cohort did reflect the majority of older Chinese [ Finally,In conclusion, this large population-based study highlighted the fact that disability, regardless of level, increased the risk of death in older adults, especially those who already suffered from chronic diseases. Moreover, compared with the old-old group, the young-old group had a lower mortality rate but a higher risk of death when ADL function disabled occurred. Health management strategies need to focus on initiatives to personalize health care and adapt to the long-term service demand of elderly with different disability statuses. In addition, interventions that prevent disability are needed to reduce the risk of death among the elders.Additional file 1: Supplement Figure S1. The Kaplan-Meier survival curve by age group and disability status.


Spatial cognitive abilities, including mental rotation (MR) and visuo-spatial working memory (vsWM) are correlated with mathematical performance, and several studies have shown that training of these abilities can enhance mathematical performance. Here, we investigated the behavioral and neural correlates of MR and vsWM training combined with number line (NL) training. Fifty-seven children, aged 6–7, performed 25 days of NL training combined with either vsWM or MR and participated in an Electroencephalography (EEG)-session in school to measure resting state activity and steady-state visual evoked potentials during a vsWM task before and after training. Fifty children, aged 6–7, received usual teaching and acted as a control group. Compared to the control group, both training groups improved on a combined measure of mathematics. Cognitive improvement was specific to the training. Significant pre-post changes in resting state-EEG (rs-EEG), common to both training groups, were found for power as well as for coherence, with no significant differences in rs-EEG-changes between the vsWM and MR groups. Two of the common rs-EEG changes were correlated with mathematical improvement: (1) an increase in coherence between the central frontal lobe and the right parietal lobe in frequencies ranging from 16 to 25 Hz, and (2) an increase in coherence between the left frontal lobe and the right parietal lobe ranging from 23 to 25 Hz. These results indicate that changes in fronto-parietal coherence are related to an increase in mathematical performance, which thus might be a useful measure in further investigations of mathematical interventions in children. Early mathematical abilities predict not only later mathematical achievement but also general school performance  and lateOne of the potential cognitive bases for math performance is spatial abilities, such as mental rotation (MR)  and visuAlthough informative of the relative benefits of vsWM and MR training, the studies by In addition, the training effect was evaluated with a steady-state evoked potentials (SSVEP) task. SSVEP has a high signal to noise ratio , which mSecondly, this study evaluated the transfer of vsWM or MR to non-trained vsWM or rotation tasks. Since vsWM and MR are positively correlated , and botLastly, we evaluated the effect sizes of these two types of spatial training combined with mathematical training on mathematical performance. A recent study suggests that different forms of spatial training combined with mathematical training have comparable effects on improving mathematics ; howeverChildren were recruited from pre-school and first-grade classes at two schools in Stockholm. Training of vsWM and MR was randomized within classes. Two training groups consisted of 63 children. Six children dropped out because of sickness. A control group was recruited from a separate school, consisting of 48 children (two children dropped out because of sickness). The demographic information for each group is shown in This work was approved by the Swedish ethical committee in Uppsala (2018/2155-31 and 2019/06514). Each child completed cognitive and mathematical tests 1–2 weeks before the training. The tests were administered in a quiet room within the schools. When a child entered the room, a tester repeated information from the written consent. Once oral consent was obtained, each child was guided to complete three mathematical tests, a vsWM test, and an MR test. This took about 15–20 min. The children in the training groups attended an additional rs-EEG recording and an SSVEP task of vsWM after a short break. The same tests were administered after training.The training was administered through an application on iPad. The children in training groups were asked to aim to do 25-session daily training at school. Each session took about 30 min. The training was delivered as a part of the daily curriculum, and the teachers were responsible for monitoring the training process. A research assistant attended the first few sessions to support the teachers. The control group attended school as usual but without the training.Due to the impact of COVID-19, changes were made during the training. While the schools remained open during the crisis, some children preferred staying at home and were willing to continue their training plans. Hence, the children were provided with hardware and software to continue. Their parents were instructed to use the application.1. Vektor aims to improve mathematical performance in children through enhancing number sense and cognitive abilities underpinning mathematical performance. Screenshots for different types of training in Vektor are shown in The training was delivered via a free app -- Vektor -- from the non-profit foundation Cognition MattersThe mathematical training included both tasks using the numberline and a task called numberpals. During NL training, children needed to solve problems by dragging along an NL. At the beginning level, an NL was presented along with an Arabic number, and children were guided to use index fingers to drag the NL from zero to the correct position corresponding to the displayed Arabic number. After a few correct trials, the Arabic number was replaced by a mathematical problem of addition or subtraction. To solve addition problems, for example, 2 + 1, children were guided to drag along the NL from zero to 2 and then from 2 to 3. For subtraction problems, e.g., 2–1, children were guided to drag along the NL to 2 and then from 2 to 1. There was no time constraint for each trial.Numberpals training was based on the ten-pal task . A bar wVisuo-spatial working memory training was conducted as previously described . SeveralMental rotation training consisted of problems in which children were expected to choose one out of three optional shapes to fit in a cut-out image. Children were guided to select the correct shape and to drag it into the cut-out part of the image. The difficulty was adaptive by increasing the degree to which the items were rotated and the complexity of the items.Three mathematical tests were administered: addition, subtraction, and verbal arithmetic. In the addition and subtraction tests, children were guided to answer arithmetic problems presented on paper by pointing at the number on cards ranging from 0 to 9. Answers larger than nine were responded by pointing twice. The test ended after 3 min or three incorrect answers in a row. Practice trials were given before formal testing.A subtest from the WISC-V was used for testing verbal arithmetic. A tester read out arithmetic problems, and the children responded verbally. Each answer was required to be given within 30 s. The test ended after three incorrect answers in a row.The spatial span board from WISC-V was used for assessing the capacity of vsWM. Children were instructed to repeat the sequence of the positions which a tester just pointed at on a span board. There were 2 practice trials. The formal test started with 2 positions and increased with 1 more position each time. Children had 2 attempts at each level, and the test would stop until 2 failures at the same level.A child-friendly MR test was used, consisting of 16 animal pictures presented on paper . At eachEEG signals were acquired via a wireless EEG system with dry sensors . Eight positions of the 10–20 system were employed to record brain activity over frontal and parietal regions, including F3, Fz, F4, P3, Pz, P4, P7, and O1. Two extra channels were used to record the vertical electrooculogram of each eye. All electrodes were online referenced to the Common Mode Sense and the Driven Right Leg attached to the right earlobe. EEG was amplified with a bandpass of 0–125 Hz and was digitized online at a sampling rate of 500 Hz.rs-EEG data were recorded during an eyes-closed resting state for 3 min. The SSVEP-WM task outline is shown in Z score of each math task. The Z scores for both pre-and post-training were based on means and standard deviations at pre-training.Because some children solved all mathematical problems within the time limit at the post-training testing – a “ceiling effect,” we calculated an efficiency measure by dividing the number of correct answers by the time spent. For synthesizing the efficiency measure from each math task, a composite score was calculated by averaging the A “ceiling effect” also existed for the post-training MR testing. Hence, an efficiency measure was calculated, indicating the number of correct answers per minute. In contrast, as no one reached the “ceiling” of the vsWM testing, the maximum WM level was used directly.EEG data were pre-processed in EEGLAB . All chaFor rs-EEG, continuous EEG data were segmented into 2-s epochs. Hence, the frequency resolution in this study was 0.5 Hz. We calculated spectrum power and coherence between 1 and 25 Hz with a step of 0.5 Hz over the fronto-parietal channels. The process was done with functions in Fieldtrip . A FouriGxy(f) is the cross-spectral power between two electrodes and Gxx(f) andyyG(f) are the two corresponding auto-spectral power. Coherence was calculated for the following 11 pairs: F3-P3, F3-Pz, F3-P4, Fz-P3, Fz-Pz, Fz-P4, F4-P3, F4-Pz, F4-P4, F3-F4, and P3-P4. Coherence values were transformed using Fisher’s z-transform.where For SSVEP-vsWM, the items to be remembered were presented in the right visual field. The analysis of SSVEP-WM focused on the contralateral occipito-parietal region. Specifically, this study quantified the response of electrodes over left posterior regions, including P3, P7, and O1, given that these regions show electrophysiological response during the vsWM delay phase e.g., . In termThe effects of the training protocols on the behavioral measures were examined by the residualized model  – the po(1)“Post vsWM ∼ Pre vsWM + Group + Age + Gender.”(2)“Post MR ∼ Pre MR + Group + Age + Gender.”The days between the pre-training testing and the post-training testing varied between children, which resulted in that children received a different amount of formal math education between the gap. Given this may affect the post-training math score, math schooling hours were estimated by the product of the days and daily math learning duration. Thus, the residualized model for math was(3)“Post math ∼ Pre math + Group + Age + Gender + Math schooling hour.”d.In the case of a significant Group effect, the planned contrasts were conducted to compare each training group to the control, and the effect size was estimated by Cohen’s The residualized model relies on the assumption that the factor of interest is not significantly correlated with covariates . Hence, t-tests with cluster-based permutations . We also examined if the pre-post differences of EEG interacted with the training protocols. The analysis began with calculating the pre-post differences in the EEG measures for the two training groups. Then, the differences were examined by the same cluster permutation approach but with two-sided independent t-tests to simulate a 2 by 2 comparison.The pre-post differences in rs-EEG power and coherence in the two training groups were examined by two-sided dependent utations . In the r, estimated from 1,000 iterations.The next step of the analysis was to examine if the pre-post EEG differences were associated with behavioral changes. The analysis was conducted by running bivariate correlations between the pre-post EEG differences that survived from the permutation tests and the pre-post differences of the behavioral tasks. The bias corrected and accelerated bootstrap 95% confidence intervals are reported together with Pearson’s t-test on LP-SSVEP.The pre-post differences in SSVEP-vsWM were examined with the paired-samples F = 0.314, p = 0.731), gender , or the math schooling hours . Also, there were no significant differences between the groups on the pre-tests .The demographic information for each group is shown in F = 5.972, p = 0.004, p = 0.028, MR + NL vs. Control, p < 0.001). The results of the planned contrasts are summarized in There was a significant effect of Group on the post-math score , whereas the MR + NL group did not  (Group had a significant effect (pre-vsWM . In the = −0.10) .F = 20.940, p < 0.001, p < 0.001, d = 1.55), and the vsWM + NL group did not .Group also showed a significant effect (e pre-MR . The plap = 0.017) and the other one ranging from 17.0 to 17.5 Hz (p = 0.023). These power differences did not interact with the training protocols (vsWM + NL vs. MR + NL), i.e., these test–retest changes were similar in both training groups.EEG analysis was conducted only in the two training groups. The results of cluster-based permutations for the effects of Session (pre-training vs. post-training) on power and coherence are shown in p = 0.001), Fz-P3 coherence ranging from 14.0 to 25.0 Hz (p = 0.002), Fz-P4 coherence ranging from 16.0 to 25.00 Hz (p = 0.006), and F3-P4 coherence ranging from 23.0 Hz to 25.0 Hz (p = 0.022). These coherence differences did not interact with the training protocols (vsWM + NL vs. MR + NL).For rs-EEG coherence, four clusters showed increased coherence at the post: P3-P4 coherence ranging from 12.5 to 23.5 Hz , i.e., no significant changes from pre- to post-training.For SSVEP-vsWM, we only included the children who completed the tasks with an accuracy above 85%. Forty-seven children met the criterion. There was no Session effect on LP-SSVEP  and the F3-P4 change   .As the two types of spatial enhancement were shown in different training groups, we examined the correlation of the EEG differences with the vsWM enhancement only in the vsWM + NL group and the MR enhancement only in the MR + NL group. There were no EEG changes correlated with the vsWM enhancement and the MR enhancement.Here, we analyzed improvements in mathematical and spatial abilities in two training groups, compared to a control group. We found that both groups improved on the pre-post tests on a combined measure of mathematical performance, compared to our control group, which is in line with prior studies of this method . In contg = 0.96, g = 0.47, Both training groups showed improvements on the trained tasks: compared to the control group, vs-WM training and MR training improved on an untrained vs-WM task and an untrained MR task, respectively, which is in line with previous findings . Our parpost hoc power calculation suggests that we had 80% power to detect an effect size of 0.69. However, a review estimated an effect size of 0.55 for transfer across different aspects of spatial tasks , and the effect of further transfer might be even smaller . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.D-WZ and TK were responsible for the study concept and design. D-WZ and AZ contributed to the data collection and data analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the IHT group, participants followed the IHT routine three times a week under hypoxia (FiO2 = 16.5%) at lactate threshold intensity. The control group (N) lived and trained under normoxia. The results showed that the 3-week LH-TL method significantly improved all lipid profile variables. The LH-TL group showed a significant increase in HDL-C by 9.0% and a decrease in total cholesterol (TC) by 9.2%, LDL-C by 18.2%, and triglycerides (TG) by 27.6%. There were no significant changes in lipid profiles in the IHT and N groups. ∆TG and ∆TC were significantly higher in the LH-TL group compared to the N group. In conclusion, hypoxic conditions combined with a mixed diet can induce beneficial changes in lipid profile even in highly trained athletes. The effectiveness of the hypoxic stimulus is closely related to the hypoxic training method.This study aimed to analyze the effects of live high-train low method (LH-TL) and intermittent hypoxic training (IHT) with a controlled mixed diet on lipid profile in cyclists. Thirty trained male cyclists at a national level with at least six years of training experience participated in the study. The LH-TL group was exposed to hypoxia (FiO Exposure of the human body to hypoxic conditions, both at rest and in combination with physical exercise, contributes to the activation of numerous adaptive mechanisms. With these adaptive changes, an increase in the effectiveness of conventional training methods is observed, resulting in improvements in athletes’ exercise capacity ,2. In reBlood lipid profile is one of the indicators of cardiovascular disease (CVD) risk. Increases in blood levels of triglycerides (TG) and LDL cholesterol (LDL-C) and decreases in HDL cholesterol (HDL-C) are associated with an elevated risk of CVD . Most stRegular physical activity improves the blood lipid profile  and atheThe lipid profile of athletes can be altered by diet, supplementation ,25,26, a2 of 14–12% interrupted by 3–5-min periods of normoxia and repeated for 60–80 min). However, it remains unclear whether the use of hypoxia during prolonged passive exposure  and training (IHT) can alter the lipid profile in individuals with high levels of training adaptation.Currently, the most common variants of altitude/hypoxic training include the live high-train low (LH-TL) method and intermittent hypoxic training (IHT) ,36,37. TWe hypothesize that the combination of hypoxia and physical exercise may contribute to beneficial changes in lipid profile and atherogenic indices in athletes. Therefore, the aim of this study was to analyze the effects of LH-TL and IHT training with a mixed diet on lipid profile and values of atherogenic indices  in trained cyclists.n = 10; age: 22.9 ± 6.3 years; body height 1.83 ± 0.08 m; body mass: 70.4 ± 7.1 kg; body fat content: 6.9 ± 2.1%; fat free mass: 65.5 ± 6.0 kg; training experience: 6.5 ± 1.3 years) was exposed to normobaric hypoxia  at rest and during sleep for 11 to 12 h a day. Training in this group was performed under normoxia. In the second experimental group (IHT) , participants followed an IHT routine three times a week under normobaric hypoxia . The control group (N)  lived and trained under normoxic conditions. Lipid profiles were not taken into consideration during the subjects’ selection. The study examined thirty trained male cyclists at a national level. The basic inclusion criteria were a minimum of six years of training experience and at least a six-month washout period from previous altitude training. All athletes had current medical examinations, without any contraindications to performing exhaustive exercise in a hypoxic environment. The participants provided their written voluntary informed consent before the participation. Study participants were randomized to three groups: two experimental groups and a control group. During the experiment, one participant from the control group withdrew from the study due to an injury. The first experimental group (LH-TL) (The research project was conducted according to the Helsinki Declaration and was approved (No. R-I-002/325/2019) by the Bioethics Committee of the Medical University of Bialystok, Poland.2 = 16.5%, ~2000 m) in a hypoxic chamber . The participants from the LH-TL and N groups followed the same training routine as the IHT group but under normoxic conditions. In addition, in the LH-TL group, participants spent 11–12 h a day (evenings and nights) under normobaric hypoxia (FiO2 = 16.5%) in a hypoxic training center .The evaluation included two research series . All the tests were performed during the pre-competitive period. Between S1 and S2, athletes from all groups followed a similar training program for 3 weeks. The only factor that differentiated the training protocols between groups was the exposure of the IHT group to normobaric hypoxia  or under hypoxia . The warm-up during all laboratory sessions was performed using the intensity at the level of 65–70% WRLT/WRLThyp. In the main part, the intensity was increased to 100% WRLT/WRLThyp. This level of intensity was maintained for 30 min (first week), 35 min (second week), and 40 min (third week). The cool-down included 15 min of continuous exercise with an intensity of 65–70% WRLT/WRLThyp. After completion of the final part of the laboratory session, cyclists performed a two-hour ride under normoxic conditions at an intensity of 60–75% WRLT.The training program included three microcycles (3 weeks) with progressive training loads. All the groups followed the same training routines with individually adjusted intensity zones . Each trCastelli’s risk index I (CRI-I) = TC/HDL-CCastelli’s risk index II (CRI-II) = LDL-C/HDL-C10(TG/HDL-C)Atherogenic index of plasma (AIP) = logDuring the experiment, each research series (S1 and S2) was performed after an overnight fast and started by drawing venous blood (10 mL) from the antecubital vein to determine total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) . The low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula: LDL-C = TC– (TG/5+HDL-C) . Furthermore, the atherogenic index (AIP) and lipid ratios  were calculated using the following formulas :Castelli2max  and lactate threshold workload (WRLT). These data were used to determine an individual training workload for the experiments.After obtaining the blood samples, body height, body mass, and body composition were also measured . Next, two hours after a light mixed meal , study participants performed a graded exercise test (40 W/3 min) using the Excalibur Sport cycle ergometer  in order to measure VOLThyp).Furthermore, after 48 h of rest during S1, the IHT group performed the same exercise test on the cycle ergometer under normobaric hypoxia conditions in order to determine the individual training load for the IHT workouts , and the baseline levels of the dependent variables were used as covariates. The post-hoc Tukey test was used for significant differences. Effect sizes (ESs) were calculated from standardized differences . Threshold values for Cohen ES statistics were considered to be small (0.20–0.60), moderate (0.60–1.20), large (1.20–2.0), very large (2.0–4.0), or extremely large (>4.0) [The data were analyzed using StatSoft Statistica 13.3 software. The results were presented as arithmetic means (x) ±standard deviations (SD). The statistical significance was set at e (>4.0) .p < 0.05; d = 0.47) and a decrease in TC by 9.2% , LDL-C by 18.2% , and TG by 27.6%  , CRI-I , and CRI-II   . There w = 0.96) . There wp < 0.05). Additionally, the analysis showed an effect of group on ∆TC and ∆AIP slightly above the accepted level of significance . ∆TG and ∆TC were significantly (p < 0.05) higher in the LH-TL group compared to the N group (p < 0.08) (p < 0.01), ∆LDL-C , ∆TG , ∆CRI-I , and ∆CRI-II . When the baseline levels of TC, LDL-C, TG, CRI-I, and CRI-II were higher, the decrease in these variables after the intervention was also higher.There was a significant effect of group on ∆TG after controlling for the effect of baseline level of TG  . The covBody mass and body composition did not change significantly with training in any group .To the authors’ knowledge, this is the first study to examine the effects of LH-TL training and IHT with a controlled mixed diet on lipid profile and atherogenic indices in athletes. The results of our study showed that a three-week LH-TL protocol  resulted in a significant improvement in all lipid profile variables analyzed and atherogenic indices. These changes were not observed in the IHT group  and in the control group of cyclists training and living in normoxia. It is common that physical activity leads to an improved lipid profile , while aThe mechanisms responsible for the improvement of the lipid profile induced by hypoxia have not yet been fully explained. Tin’kov and Aksenov  suggesteThe mechanisms associated with the improvement of HDL-C after LH-TL are also unclear. In a recent study, Gao et al.  suggeste2max) at 2000–2500 m [2 = 15%, ~3000 m) reduced blood TG levels in healthy male non-athletes, which was not observed after training under normoxia. IHT training sessions were performed three times a week at an intensity around the lactate threshold. The findings were not confirmed by our results. After a training protocol similar to that used in the study of Haufe et al. [2 = 16.5%, ~2000 m). There are reports in the literature that not only prolonged exposure to hypoxia but also regular training sessions under these conditions have a beneficial effect on lipid profile. Haufe et al.  suggeste0–2500 m ,43,45. I0–2500 m  observed0–2500 m  demonstre et al. , no signWe can presume that the intensity of the hypoxic stimulus may be a key factor in the beneficial effects of IHT on lipid profile. Furthermore, it is important to note that the intensity of hypoxia induced by IHT results from the combination of two components: altitude and exercise intensity. Wood et al.  reportedThe best recognized environmental factor that can significantly modify the lipid profile is the diet. For example, a recent meta-analysis revealed that dietary fat manipulation has a significant influence on blood lipid levels in people with overweight or obesity without metabolic disturbances . ResultsThe results of this study indicate that the extent of changes in lipid variables is related to baseline values by the following rule: the higher the baseline level of TC, LDC-C, TG, CRI-I, and CRI-II, the greater their improvement in response to hypoxia. Some studies reveal that athletes who consume high-carbohydrate or high-fat diets increase their risk of developing hypercholesterolemia. In particular, this phenomenon was observed in keto-adapted endurance athletes  and athlOur findings indicate that the effectiveness of the hypoxic stimulus is closely related to the training method using the hypoxic environment, which determines the duration of exposure of the human body to hypoxia. Our results provide evidence of the beneficial effects of hypoxia rather than just exercise or diet on the lipid profile in trained individuals. The most beneficial changes in lipid profile were observed in the LH-TL group, which was passively exposed to hypoxia for the longest time (11 to 12 h a day for 3 weeks). Similar training with a controlled mixed diet in the control and IHT groups did not induce similar adaptations. Based on the results of this study, it can also be concluded that the magnitude of changes in the lipid profile after the hypoxic intervention will be greater in athletes with a disturbed lipid profile.